Background: P. aeruginosa exhibits several efflux pump systems that allow it to be resistant to several antimicrobial agents. Phenylalanine arginyl β-naphthylamide (PAβN) is an Efflux pump inhibitor (EPI) that can inhibit several multidrug efflux pumps. Objective: This study aimed to detect the efflux pump activity in FQ resistant P. aeruginosa, and to investigate the role of PAβN on FQ resistance. Methodology: P. aeruginosa isolates were subjected to antibiotic susceptibility testing by disc diffusion and those resistant to ciprofloxacin and levofloxacin were subjected to MIC detection before and after addition of PAβN. Also reverse transcription RT PCR was done for detection of mexA, mexC, mexE and mexX genes overexpression in the FQ resistant strains. Results: After the addition of PAβN, 95.9% and 94.5% of the isolates showed reduction in MICs of ciprofloxacin and levofloxacin respectively, 8.20% and 27.4% of the isolates restored susceptibility to those drugs respectively and 8.20% reverted to levofloxacin intermediate breakpoint. MexE was the most common efflux pump overexpressed, followed by mexX and mexA. When using EPI 96.8% and 95.3% of the isolates showed both overexpression and reduction of ciprofloxacin and levofloxacin MIC respectively. Using a cut- off point of ≥4 fold reduction in levofloxacin MIC discriminates efflux pump overexpressing from non-overexpressing isolates, (sensitivity of 70%, specificity of 67%). Conclusion: Efflux pump mediated resistance is an important mechanism contributing to multidrug resistance in P.aeruginosa as proven phenotypically and genotypically, with mexE being the most commonly expressed. The addition of PAβN to levofloxacin could be used as a phenotypic test for detection of efflux pump overexpressing isolates and may be effective to restore the levofloxacin susceptibility.